Studies On Fermentation Technology Of Porcine Lactoferrin Gene-engineering Strain

Lactoferrin (LF) is a multifunctional glycoprotein in physiological fluids of mammals. Several functions have been proposed for lactoferrin, including protection against microbial infection, regulation of immune function, regulation of myelopoiesis, cellular growth promotion, regulation of intestinal iron absorption and regulation of transcription. This paper was written based on the success of the porcine lactoferrin gene cloning and expression in P. methanolica system by our group, the study was conduct to research the fermentation technology of porcine lactoferrin gene-engineering strain, and to evaluate the value of fermentation product on anti-bacteria activity. The results were summarized as follows:Firstly the growth rhythm of recombinant strain was studied; the strain went through lag phase, logarithmic phase, deceleration phase and stationary phase during in growing 20 hours. The time when the strain growth was at the middle of logarithmic phase (13 hours) in seed culture medium was selected as seed for future study. The expression rule of target protein was also studies in 168 hours fermentation when methanol was used for lactoferrin expression. The result showed expression capacity of latoferrin was enhance first, and restrained at 144-hour later.Single factor experiment was used to find the most suitable carbonic source, the most suitable nitrogen source, addition level and C/N. After single factor experiment, we used orthogonal combination experiment design to choose the optimum fermentation medium for lactoferrin expression, and we also studies the related affecting key factors such as initial pH, inoculation volume on lactoferrin expression. The favor culture medium in shake flask:carbon source (potato starch) 40g/L,nitrogen source (soybean) 8g/L,biotin 4ml/L (4×10-5%),MgSO4.7H2O 5g/L and KH2PO4 3g/L. With 50mL medium in 250mL flask temperature 30℃shaker rotate speed as 250 r/min,pH=4.4 (at the early stage)/5.0 (later) and inoculation volume 4% were accessed as the best condition for shaking flask fermentation, and under the optimum conditions the expression of target protein was 55.5μg/mL by reversed-phase high performance liquid chromatography.Based on the results in shake flake, a 10L fermenter was used to evaluate the growth and target protein expression of gene-engineering strain with batch Fermentation. The results proved the culture medium which was screened in shake flake could be well used for growth and lactoferrin expression of the gene-engineering strain. With fed-batch fermentation study in 10L fermenter, the results showed the basic metabolism rule and the related fermentation technology of recombinant strain. The optimal technological parameters in 10L fermenter:agitation speed was 300-350 r.min-1(sectional control),the optimal temperature was 28-30℃(sectional control),the optimal initial pH was 7.5, after 24 hours fermentation addition methanol into fermenter every 12-hour. The lactoferrin quantitative analysis of 10L sample by reversed-phase high performance liquid chromatography was 604.4μg/mL.At last, the value of fermentation product on anti-bacteria activity was also studied with agar diffusion test and time-kill method. The fermentation product had a direct effect on the growth inhibition of E.coli K88 in agar diffusion test experiment, and the inhibition on E.coli K88 had a direct relationship with lactoferrin level. In addition, the time-kill method gave a developing inhibition and killing process of fermentation product on E.coli K88, salmonella typhimurium and salmonella choleraesuis.The study of this paper is based on industrialization application, the culture medium for industrial fermentation screened out in shake flake experiment, and the culture medium could be used for strain growth and protein expression in 10L fermenter. The related technical parameters which were collected in shake flake or 10L fermenter will be useful in future fermentation application. In addition, the antibacteria tests of fermentation product also provide evidence for ferment-lactoferrin use as the substitutes of antibiotics.